Objectives: Nanoparticles (NPs), with their sophisticated contrast, have become valuable tools as molecular imaging agents for providing specific cellular targeting and sensitive localization. Among various NP types, gold NPs stand out with unique features such as chemical stability, biocompatibility, adjustable shape, and size-dependent optical properties, making them particularly promising for molecular detection by leveraging surface-enhanced Raman scattering (SERS)1. This effect utilizes the inelastic scattering of light to generate unique spectral signatures, which can be further augmented by a plasmon resonance phenomenon induced by the NP’s spherical gold core. Various batches of SERS NPs have been fabricated, each embedded with a unique Raman active layer capable of generating a distinctive spectral barcode that can offer unprecedented multiplexed Raman imaging capabilities2. The large surface area of the NP also allows for further conjugation with a fluorophore for fluorescence imaging. The challenge lies in ensuring the effective targeting of specific cells and biomarkers by our multimodal NPs amidst the complexity of diverse cell types and biomolecules, requiring high specificity. Here, we develop an optimized protocol to improve targeting efficiency of our multimodal NPs in cellular applications. SERS NPs, capable of providing contrast using both fluorescence and Raman imaging are conjugated with antibodies and incubated with cancer cells overexpressing relevant proteins. Their specific binding potential is then quantified using flow cytometry to achieve optimal targeting efficiency. Next, fluorescence and Raman imaging techniques are employed together to assess the NPs’ ability to efficiently target suspended cells. This study underscores the pivotal role of multimodal NPs in effective active targeting, making them invaluable for multiplexing applications across diverse biological environments.
Methods: Multimodal NP Preparation: SERS NPs were synthesized by a Raman reporter molecule being adsorbed to a gold core’s surface, silica coated, thiolated, conjugated with DyLight 650 fluorophores and biomarker targeting antibodies (Fig. 1a). Characterization of Multimodal NPs: NP size and charge were measured during synthesis with concurrent assessment of thiols, fluorophores, and antibodies per NP. Specific Binding Evaluation: Cancer cells (~200,000 cells) were incubated with 20 µL of 1.5 nM conjugated NPs for 15 min at 4°C on a shaker and measured with flow cytometry. Cell Imaging: Cells were incubated with bioconjugated NPs targeting HER2 and plated on a 12-well adhesion slide.
Results: We demonstrate the consistency of multimodal NPs, incorporating Raman reporters, fluorescent dyes, cross-linkers, and specific antibodies, across multiple batches to ensure the robustness of our method (Fig. 1a). We found that achieving 300 antibodies per NP was crucial for effective binding, demonstrating significant specific-to-nonspecific binding (p<0.0001) (Fig. 1b). Using flow cytometry, we determined the optimal NP concentration to be 18,000 NPs per cell for suspended cells (Fig. 1c). Environmental conditions, such as 15-minute stain at 4°C on a gentle shaker, and the length of the cross-linker on NPs were also crucial, with SM(PEG)12 showing significantly greater specific-to-nonspecific binding (persuasive data, Fig. 1d). Fluorescence and Raman imaging confirmed the high specific binding capacity of NP conjugates using the optimized staining parameters, revealing significant differences between negative and positive HER2-expressing cancer cells (Fig. 1e).
Conclusion: We established a reliable and robust protocol for conjugation and cellular incubation to optimize NP binding to a range of biological markers across various cancer cell lines. We developed a multimodal approach for identifying cancer markers, leveraging the precise biomarker distribution mapping. These findings hold significant promise for various multiplexed molecular profiling applications offering enhanced insight into a patient’s tumor and its unique molecular expression profile. This highly multiplexed platform offers a noninvasive avenue for cancer diagnosis, classification, and for analyte detection and characterization in the clinical setting.
Image/Figure:
Click to View
Image/Figure Caption:
Figure 1. Achieving Maximum Targeting Efficiency of Cell Surfaces with Actively Targeted SERS Nanoparticles.
(a) (A) Labeling of gold nanoparticles (NPs) with BPE Raman reporter, then silica-coated and thiolated with TEOS and MPTMS, respectively. SERS NPs were then fluorescently labeled with DyLight650-maleimide and bioconjugated with a specific monoclonal antibody. Remaining thiols are capped on the surface of NPs and centrifuged for purification prior to application. Active targeting by the prepared multimodal NPs was evaluated with cancerous cell lines known for their protein overexpression. (B) Quantification of (i) thiols per NP, (ii) NP size, (iii) zeta potential, (iv) number of fluorophores per NP, and (v) antibodies (Ab) per NP.
(b) (A) (i) Digital photo of NP samples following the addition of immunoglobulin G (IgG) and epidermal growth factor receptor (EGFR) at varying antibody (Ab) per nm2 or NP ratio. The red circles highlight the NP precipitation when adding more Ab per nm2. (ii) Evaluating different Ab concentrations following a 12 h shake with the capping reagent, MM(PEG)12. (B) (i) Reaction yield of measured Ab/NP versus how much Ab actually binds to the NP. The blue line represents IgG-conjugated NPs while the red line shows EGFR-conjugated NPs with different Ab/nm2 ratios. (ii) Mean fluorescence intensity (MFI) of flow cytometry results using A431 cells. The outcomes for NPs with 0 Ab/103 nm2, IgG, and EGFR conjugations are depicted through gray, blue, and red bars, respectively.
(c) (A) NPs actively targeting cell surface markers of A431 cells. (B) Ratio of specific-to-nonspecific binding at various staining concentrations of NPs/cell. (C) Flow cytometry findings from the addition of different multimodal NP quantities to suspended A431 cells. Yellow peaks represent unstained cells, blue peaks correspond to cells stained with NPs conjugated to the isotype control (IgG), and red peaks correlate with cells stained with NPs conjugated to EGFR antibody.
(d) (A) Influence of different cross-linker lengths (3.5, 5.2, and 9.5 nm) on NPs was examined alongside an isotype control and an anti-EGFR antibody. (B) Flow cytometry plots of NPs conjugated with SM(PEG)6 (3.5 nm), SM(PEG)12 (5.2 nm), and SM(PEG)24(9.5 nm) that are conjugated with IgG and EGFR antibodies for their targeting efficiency. Yellow peaks represent unstained cells, blue peaks correspond to cells stained with NPs conjugated to IgG, and red peaks correlate with cells stained with NPs conjugated to EGFR. (C) Quantitative ratiometric analysis of specific EGFR NP binding to nonspecific isotype IgG NP. SM(PEG)12, which has a length of 5.2 nm, showed the greatest binding ratio compared to SM(PEG)6 (3.5 nm) and SM(PEG)24 (9.5 nm).
(e) (A) White light, fluorescence, and Raman images of HCC70 and SKBR3 cells when incubated with IgG or HER2-targeted NPs. These cells are in suspension and fixed with a coverslip prior to imaging. The left side of each panel is a white light image; the middle represents a fluorescence image, and the right side is a Raman image. Scale bars are represented by 50 μm. (B) MFI values and ratiometric quantifications of HCC70 and SKBR3 cells incubated with conjugated IgG (blue) and HER2 (green)-conjugated NPs. (C) Raman intensity and ratiometric assessment in HCC70 and SKBR3 cells when incubated with NPs conjugated with IgG (blue) and HER2 (green). There is a significant difference between IgG and the overexpressing biotargets in SKBR3 cells. ****p < 0.0001, ***p < 0.001, **p < 0.01. All error bars represent the standard error of the mean.
Author
University of Southern California